Currently, many attempts have been conducted to utilize a protein solution as liquid droplets. For example, for the drug delivery method, the liquid droplets have been considered to be applied in transmucosal administration because of, for example, advantages in that only a small amount of protein may be required in the production of a biochip or biosensor and the protein may be integrated easily. In addition, attentions have been paid to a method of using a fine liquid droplet of protein for control on crystallization of protein and also for screening of a physiologically active substance (see, for example, Japanese Patent Application Laid-Open No. 2002-355025, Allain L R et al., “Fresenius J. Anal. Chem”, vol. 371, p. 146-150, 2001, and Howard E I and Cachau R E, “Biotechniques”, vol. 33, p. 1302-1306, 2002).
In recent years, mass production of proteins, particularly useful proteins such as enzymes and those having physiological activities has become possible by any technology such as genetic recombinant technology. Therefore, the process of making protein into liquid droplets can be an effective means in the field of searching, utilizing, and applying a novel protein medicine. More specifically, there are increasing significant demands on means for providing patients with many pharmaceutical agents by microdroplets. In particular, microdroplets have become important for the administration of proteins, peptides, and other biological materials from the lungs. In other words, the lungs have been remarked as an administration route in place of an injection of a macromolecule peptide-based drug represented by insulin because the lungs have lung alveolis with their own extensive surface areas of 50 to 140 m2 and the epithelium provided as a barrier of absorption is as thin as 0.1 μm, while the enzyme activities of the lungs are smaller than those of the gastrointestinal tract.
In general, the deposition of microdroplets of drug in the lungs has been known to depend largely on the mass median aerodynamic diameters thereof. In particular, the delivery of the microdroplets to the lung alveolis in the deep portions of the lungs essentially requires the development of: a stable pharmaceutical preparation which can be given with high reproducibility for a narrow particle size distribution of 1-5 μm of the droplets; and an administration form.
As a method of preparing uniform droplets with a narrow particle size distribution, the use of a suitable droplet generator diverted from those used in inkjet printing in the production of extremely fine droplets and the application of the droplets have been reported in the art (see, for example, U.S. Pat. No. 5,894,841 and Japanese Patent Application Laid-Open No. 2002-248171). Here, the specific inkjet printing method concerned involves leading liquid to be ejected into a small chamber where the liquid is subjected to an ejection pressure, thereby allowing droplets of the liquid to be ejected from orifices. A discharging method may be any one of those known in the art, such as one that generates air bubbles spouting droplets through orifices formed on a chamber by means of thermal transducers such as thin-film resistors (i.e., a thermal inkjet method) and one that ejects liquid directly from orifices formed on a chamber by means of piezoelectric transducers (i.e., a piezo inkjet method). The chamber and the orifices are incorporated in a print head element and the element is then connected to both a liquid-supplying source and a controller for controlling the ejection of droplets of the liquid.
For allowing the lungs to absorb a drug, the dose of the drug should be controlled. Therefore, making droplets from the liquid by the inkjet method, which is capable of adjusting the ejection amount thereof, is a very preferable configuration. On the other hand, however, the ejection of a solution should be surely carried out in this case. However, the ejection of a protein solution is unstable when the protein solution is only controlled with respect to its surface tension and coefficient of viscosity. Therefore, the difficulty in quantitative ejection has often occurred.
Problems associated with the process of making droplets of a protein or peptide using the inkjet method are due to the fact that the tertiary structure of the protein is brittle. Thus, the aggregation or degradation of the protein may occur when the configuration thereof is destroyed. When the process of making droplets is carried out using the inkjet method, the structures of many proteins may become unstable owing to physical stress such as pressure or shearing stress or to high surface energy peculiar to microdroplets (if the thermal inkjet method is employed, heat is further applied in addition to the above stress). In particular, in the case of the inkjet method, those physical actions are extremely larger than the shearing force and thermal energy to be applied by conventional stifling or heat treatment (for example, in the case of the thermal inkjet method, approximately 300° C. and 90 atm are applied momentarily). Furthermore, several physical stresses are impressed at the same time, so that the stability of protein may tend to be substantially lowered, compared with the case of usually handling the protein. On this account, the conventional technology for stabilizing protein may be insufficient when the inkjet technology is employed. If such a problem occurs, the protein may be aggregated at the time of making droplets and nozzles may be then clogged, thereby making it difficult to eject droplets.
Furthermore, droplets having diameters of 1 to 5 μm, which are suitable for the inhalation into the lungs, are extremely smaller than those having diameters of approximately 16 μm used in any printer commercially available at present. Therefore, a larger surface energy or shearing stress may be impressed on the droplets than on the droplets used in the printer. Therefore, it is more difficult to eject microdroplets suitable for inhaling protein into the lungs.
In consideration of the diameters of the droplets as described above, a thermal inkjet method capable of having highly-densified nozzles and a low production cost is preferably used as a method of discharging a protein solution.
In the pamphlet of International Publication No. WO 02/094342, there is disclosed a compound for regulating surface tension and a method of adding a humectant for a liquid composition for the pulmonary absorption of a droplet prepared using a thermal inkjet method. Here, for increasing the stability of protein in a solution provided as droplets by the surface tension, viscosity, or moisturizing action of the solution, a surfactant or a water-soluble polymer such as polyethylene glycol is added.
However, there is no description about the stability of discharging, and also the addition of a surfactant or a water-soluble polymer exerts insufficient effects when the concentration of protein or peptide increases. As a result, the additive itself often blocks the stability of discharging. In addition, there are more surfactants which have been recognized as of no effect at all than those having effects. Besides, the stability is defined by not only the surface tension and viscosity. Therefore, the method disclosed in the document is not a common practice for the stabilization of ejection. Therefore, for actual use, any liquid to be used for an ejection purpose, which is capable of discharging protein or peptide in a stable manner, becomes essential.
In addition, each of Japanese Patent Application Laid-Open No. 2003-154655 and Japanese Patent No. 03610231 discloses a method of preparing a protein chip or the like as a method of utilizing a protein or peptide such that it is ejected by a thermal inkjet method. However, there was no substantial description about stable ejection of the protein and peptide.
As described above, the inkjet method has been known in the art as one of the methods of discharging a liquid sample after making it into fine droplets. The inkjet method has a characteristic feature of showing high controllability for the amount of liquid to be ejected after being provided as droplets even if the amount is extremely small. Methods of discharging microdroplets based on the inkjet method include a vibration method using a piezoelectric element or the like and a thermal inkjet method using a microheater element. In the case of the vibration method using the piezoelectric element or the like, reducing the size of a piezoelectric element to be used is limited and the number of ejection orifices formed per unit area is also restricted. Besides, production costs increase as the number of ejection orifices per unit area increases. In contrast, in the case of the thermal inkjet method, the size of the microheater element can be comparatively easily reduced, and also the number of ejection orifices formed per unit area can be larger than that of the vibration method using a piezoelectric element or the like, while the production costs thereof can be also significantly reduced.
When the thermal inkjet method is applied, for controlling the state of appropriate aerosol of microdroplets of liquid to be ejected from respective orifices and the amount of the liquid, there is a need of coordinating the physical properties of the liquid to be ejected. In other words, the composition of the liquid, including the kind and composition of a solvent and the concentration of a solute, the solvent and the solute being contained in the sample of liquid to be ejected, should be schemed so as to be adjusted to obtain a desired volume of the microdroplet. Furthermore, various kinds of mechanisms for discharging droplets on the basis of the principle of the thermal inkjet method have been also developed. Concretely, for a conventional inkjet head to be mounted on a printer, the amount of each liquid droplet to be ejected is on the order of several pico-liters. While technologies of an ejection mechanism and an ejection method, which allow droplets to be formed as those having extremely small sizes in the order of several sub-pico liters or femto-liters, have been developed (see the pamphlet of International Publication No. WO 02/092813). For instance, when somatic cells of several micrometers in size are provided as a target to be coated with a drug, it is assumed that using the extremely fine droplets as individual droplets to be ejected may be requested.
There is known, as a method of stabilizing protein, a method involving adding any one of water-soluble polymers such as surfactants, glycerols, various kinds of hydrocarbons, and polyethylene glycols as well as albumin. Also, there are known, as methods of preserving a protein preparation for a long term, several methods (Japanese Patent No. 03618633, International Publication No. WO 02/017957, and National Publication No. of the translated version of PCT application 2003-510368), where amino acids are added to erythropoietin, G-CSF, and interferon. However, according to the study conducted by the inventors of the present invention, on the basis of the kind and concentration of protein or peptide or the concentration of an additive, those methods may be insufficient for the stability of ejection in the thermal inkjet method.
In many cases, there is substantially no effect on an improvement in ejection performance when protein or peptide is ejected even after formulation with polyols such as ethylene glycol and glycerin, and a humectant such as urea, which are suitable additives for ink to be used in inkjet printing. Therefore, it has been demanded to develop an ejection liquid to stably eject a protein or peptide solution on the basis of the principle of the inkjet method using thermal energy.